Method and compositions for detecting botulinum neurotoxin

ABSTRACT

A molecular construct capable of fluorescent resonance energy transfer (FRET), comprising a linker peptide, a donor fluorophore moiety and an acceptor fluorophore moiety, wherein the linker peptide is a substrate of a botulinum neurotoxin selected from the group consisting of synaptobrevin, syntaxin and SNAP-25, or a fragment thereof capable being cleaved by the botulinum neurotoxin, and separates the donor and acceptor fluorophores by a distance of not more than 10 nm, and wherein emission spectrum of the donor fluorophore moiety overlaps with the excitation spectrum of the acceptor fluorophore moiety; or wherein the emission spectra of the fluorophores are detectably different. Also provided are isolated nucleic acid expressing the construct, kits comprising said construct and cell lines comprising said nucleic acid. Further provided are methods of detecting a BoNT using the above described construct via FRET, and methods for detecting a BoNT using surface plasmon resonance imaging.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority of U.S. Provisional Application Ser.No. 60/530,645, filed Dec. 19, 2003, and U.S. Provisional ApplicationSer. No. 60/579,254, filed Jun. 15, 2004. The contents of bothprovisional applications are incorporated herein by reference in theirentirety.

GOVERNMENT INTEREST

This invention was made with United States government support awarded bythe National Institutes of Health under the grant numbers NIH GM56827and MH061876. The United States has certain rights in this invention.

BACKGROUND OF THE INVENTION

Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum andare the most potent toxins known. These toxins are a well-recognizedsource of food poisoning, often resulting in serious harm or even deathof the victims. There are seven structurally related botulinumneurotoxins or serotypes (BoNT/A-G), each of which is composed of aheavy chain (˜100 KD) and a light chain (˜50 KD). The heavy chainmediates toxin entry into a target cell through receptor-mediatedendocytosis. Once internalized, the light chain is translocated fromendosomal vesicle lumen into cytosol, and acts as a zinc-dependentprotease to cleave proteins that mediate vesicle-target membrane fusion(“substrate proteins”). Cleavage of SNARE proteins blocks vesicle fusionwith plasma membrane and abolishes neurotransmitter release atneuromuscular junction.

These BoNT substrate proteins include plasma membrane protein syntaxin,peripheral membrane protein SNAP-25, and a vesicle membrane proteinsynaptobrevin (Syb). These proteins are collectively referred to as theSNARE (soluble N-ethylmaleimide-sensitive factor attachment proteinreceptor) proteins. Among the SNARE proteins, syntaxin and SNAP-25usually reside on the target membrane and are thus referred to ast-SNAREs, while synaptobrevin is found exclusively with synapticvesicles within the synapse and is called v-SNARE. Together, these threeproteins form a complex that are thought to be the minimal machinery tomediate the fusion between vesicle membrane and plasma membrane. BoNT/A,E, and C¹ cleave SNAP-25, BoNT/B, D, F, G cleave synaptobrevin (Syb), atsingle but different sites. BoNT/C also cleaves syntaxin in addition toSNAP-25.

Botulinum neurotoxins are listed as a bioterror threat due to theirextreme potency and the lack of immunity in the population. Because oftheir paralytic effect, low dose of botulinum neurotoxin has also beenused effectively to treat certain muscle dysfunctions and other relateddiseases in recent years.

Due to their threat as a source of food poisoning, and as bioterrorismweapons, there is a need to sensitively and speedily detect BoNTs.Currently, the most sensitive method to detect toxins is to performtoxicity assay in mice. This method requires large numbers of mice, istime-consuming and cannot be used to study toxin catalytic kinetics. Anumber of amplified immunoassay systems based on using antibodiesagainst toxins have also been developed, but most of these systemsrequire complicated and expensive amplification process, and cannot beused to study toxin catalytic activity either. Although HPLC andimmunoassay can be used to detect cleaved substrate molecules andmeasure enzymatic activities of these toxins, these methods aregenerally time-consuming and complicated, some of them requirespecialized antibodies, making them inapplicable for large scalescreening. Therefore, there is a need for new and improved methods andcompositions for detecting BoNTs.

There is also a need for improved technique for screening for inhibitorsof BoNTs. These inhibitors can be used as antidotes to the toxins forboth preventive and treatment purposes.

Recently, a new approach based on intramolecular quenching offluorigenic amino acid derivatives has been explored. In principle, twoamino acid derivatives are used to replace two native amino acids in avery short synthetic peptide (20-35 amino acids) that containing toxincleavage sites. The fluorescence signal of one amino acid derivative isquenched by another amino acid derivative when they are close to eachother in the peptide. Cleavage of the peptide separates two amino acidderivatives and an increase in fluorescence signal can be detected(Schmidt J, Stafford R, Applied and Environmental microbiology, 69:297,2003). This method has been successfully used to characterize a BoNT/Binhibitor. However, it requires synthesis of peptides with modifiedamino acid derivatives and is not suitable for use in living cells.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a molecular constructcapable of fluorescent resonance energy transfer (FRET), comprising alinker peptide, a donor fluorophore moiety and an acceptor fluorophoremoiety, wherein the linker peptide is a substrate of a botulinumneurotoxin selected from the group consisting of synaptobrevin, syntaxinand SNAP-25, or a fragment thereof that can be recognized and cleaved bythe botulinum neurotoxin (“cleavable fragment”), and separates the donorand acceptor fluorophores by a distance of not more than 10 nm, andwherein emission spectrum of the donor fluorophore moiety overlaps withthe excitation spectrum of the acceptor fluorophore moiety.

Preferably, the donor fluorophore moiety is a green fluorescent proteinor a variant thereof, and the acceptor fluorophore moiety is acorresponding variant of the green fluorescent protein.

In one embodiment, the linker peptide comprises at least about 14 aminoacid residues and an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1-6. In a preferred embodiment, the linkerpeptide comprises at least about 15, or at least about 16, or at leastabout 17, or at least about 18, or at least about 19, or at least about20, or at least about 21, or at least about 22, or at least about 23, orat least about 24, or at least about 25, or at least about 26, or atleast about 27, or at least about 28, or at least about 29 amino acidresidues, and a sequence selected from the group consisting of SEQ IDNOs: 1-6.

In a preferred embodiment, the linker peptide comprises at least about30 amino acid residues and an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1-6. More preferably, the linker peptidecomprises at least about 35 amino acid residues, or at least about 40amino acid residues, or at least about 45 amino acid residues, or atleast about 50 amino acid residues. In a particularly preferredembodiment, a construct of the present invention comprises a linkerpeptide that comprises at least about 55 amino acid residues, or atleast about 65 amino acid residues.

The present invention further provides an isolated polynucleotidemolecule encoding a construct described above. The construct ispreferably an expression vector comprising the polynucleotide moleculeoperably linked to a promoter. A preferable promoter for the inventionis an inducible promoter.

The present invention also provides a cell comprises an isolatedpolynucleotide molecule described above. In one embodiment, the cell isselected from the group consisting of a primary cultured neuron cell,PC12 cell or a derivative thereof, a primary cultured chromaphin cell, aneuroblastoma cell, a human adrenergic SK-N-SH cell, and a NS-26 cellline. Preferably, the cell is a cortical neuron cell, a hippocampalneuron cell, a spinal cord motor neuron cell, or a murine cholinergicNeuro 2a cell.

In a further embodiment, the present invention provides a kit whichcomprises a construct of the present invention in a suitable container.

Also disclosed herein is a method for detecting a botulinum neurotoxin,the method comprising providing a construct described hereinabove,wherein the linker is substrate protein or a fragment thereofcorresponding to the botulinum neurotoxin to be detected, exposing theconstruct to a sample suspected of containing a botulinum neurotoxinunder a condition under which the botulinum neurotoxin cleaves theprotein substrate or a fragment thereof, and detecting and comparing theFRET signal before and after the construct is exposed to the sample,wherein a decrease in FRET indicates the presence of botulinumneurotoxin in the sample. In a preferred embodiment, additional Zn²⁺ isadded to the sample to be detected. The method of the invention issuitable for the detection of a botulinum neurotoxin selected from thegroup consisting of BoNT/A, E, and C, and the corresponding substrateprotein is SNAP-25 or a cleavable fragment thereof. The method of thepresent invention is also suitable for the detection of BoNT/B, D, F orG, using synaptobrevin (Syb) or a cleavable fragment thereof as acorresponding substrate protein. Similarly, the method of the presentinvention is suitable for detecting BoNT/C, with SNAP-25 or a cleavablefragment thereof as a corresponding substrate protein.

In a preferred embodiment, for the method of the present invention, FRETis detected by a method selected from the group consisting 1) measuringfluorescence emitted at the acceptor (A) emission wavelength and donor(D) emission wavelength, and determining energy transfer by the ratio ofthe respective emission amplitudes; 2) measuring fluorescence lifetimeof D; 3) measuring photobleaching rate of D; 4) measuring anisotropy ofD or A; and 5) measuring the Stokes shift monomer/excimer fluorescence.

A particularly preferred fluorophore pair for the present invention isCFP-YFP.

The present invention also provides a method for screening for aninhibitor of a botulinum neurotoxin, comprising providing a cellgenetically engineered to express a construct as described above,wherein the linker in the construct is a substrate peptide correspondingto the botulinum toxin; exposing said cell to the botulinum neurotoxinin the presence of a candidate inhibitor compound; and detecting FRETsignals of the cell before and after said exposing to the botulinumtoxin, wherein an observation of substantially no decrease in FRET,compared to a cell exposed to the botulinum neurotoxin in the absence ofthe candidate inhibitor, indicates that the candidate inhibitor iscapable of inhibiting the botulinum neurotoxin. Preferably, thecandidate compound is among a library of compounds and the method is ahigh throughput method.

In a further embodiment, the present invention provides a method fordetecting a botulinum neurotoxin, the method comprising depositing alayer of a BoNT target peptide onto a metal surface, exposing said metalsurface having BoNT target peptide on its surface to a sample suspectedof containing a corresponding BoNT, under conditions to allow the BoNTto cleave the target peptide on the metal surface, and measuring anydecrease in the molecular weight of the target peptide bound to themetal surface as a result of BoNT cleavage via surface plasmon resonantimaging.

Another embodiment of the present inventions is a method for detecting abotulinum neurotoxin, the method comprising, a) providing a construct,wherein the linker is a substrate protein or a cleavable fragmentthereof corresponding to the botulinum neurotoxin to be detected, andwherein the construct is anchored to a plasma membrane of cell, suchthat the linker protein adopts a conformation with which FRET occursbetween the donor and acceptor fluorophore, b) exposing the construct toa sample suspected of containing a botulinum neurotoxin under acondition under which the botulinum neurotoxin cleaves the proteinsubstrate or a fragment thereof, and c) detecting and comparing the FRETsignal before and after the construct is exposed to the sample, whereina decrease in FRET indicates the presence of botulinum neurotoxin in thesample.

The present invention further provides a molecular construct comprisinga linker peptide, a first fluorophore moiety and a second fluorophoremoiety, wherein the linker peptide is a substrate of a botulinumneurotoxin selected from the group consisting of synaptobrevin, syntaxinand SNAP-25, or a fragment thereof that is able to be cleaved by thebotulinum neurotoxin, and wherein emission spectrum of the firstfluorophore moiety is detectably different from the excitation spectrumof the second fluorophore moiety. Preferably, the linker is afull-length protein of the substrate synaptobrevin, syntaxin or SNAP-25.Preferably, the construct is anchored to a vesicle, which may or may notbe inside a cell. The present further provides a polynucleotideconstruct encoding the above polypeptide construct.

The present invention further provides a method for detecting abotulinum neurotoxin, the method comprising a) providing a peptideconstruct as described above, b) exposing the construct to a samplesuspected of containing a botulinum neurotoxin under a condition underwhich the botulinum neurotoxin cleaves the protein substrate or afragment thereof, and c) detecting spatial separation of thefluorescence signals of the first and second fluorophores, whereinoccurrence of spatial separation indicates the presence of botulinumneurotoxin in the sample. Preferably, the vesicle is inside a live cell,the linker peptide is CFP-SNAP-25 (1-197) linked to SNAP-25(198-206)-YFP, wherein detection of CFP fluorescence but not YFPfluorescence indicates the existence of presence of botulinum neurotoxinin the sample.

The invention is described in more details below with the help of thedrawings and examples, which are not to be construed to be limiting thescope of the present invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depiction of the CFP-YFP based bio-sensors formonitoring botulinum neurotoxin protease activity. FIG. 1A is a designof the bio-sensor constructs. CFP and YFP are connected via a fragmentof synaptobrevin (amino acid 33-94, upper panel), or SNAP-25 (amino acid141-206, lower panel), respectively. The cleavage sites for eachbotulinum neurotoxin on these fragments are labeled. FIG. 1B shows thatCFP and YFP function as a donor-acceptor pair for FRET, in which theexcitation of CFP results in YFP fluorescence emission (upper panel).Energy transfer between linked CFP and YFP is abolished after cleavageof the synaptobrevin or SNAP-25 fragment with botulinum neurotoxins(lower panel). The optimal excitation wavelength for CFP is 434 nM, andthe emission peak is 470 nM for CFP, and 527 nM for YFP.

FIG. 2 shows the fluorescence emission spectra of the recombinantbio-sensor proteins. FIG. 2A shows the emission spectra of therecombinant his₆-tagged CFP and YFP alone (300 nM), as well as themixture of these two proteins (1:1). The fluorescence signals werecollected from 450 to 550 nM using a PTIQM-1 fluorometer in Hepes buffer(50 mM Hepes, 2 mM DTT, and 10 μM ZnCl₂, pH 7.1). The excitationwavelength is 434 nM, the optimal for CFP. The YFP protein only elicitsa small fluorescence emission signal by direct excitation at 434 nM.FIG. 2B shows the emission spectra of recombinant his₆-taggedCFP-SybII-YFP, collected as described in panel FIG. 2A. The arrowindicates the YFP emission peak resulted from FRET.

FIG. 3 depicts that the cleavage of bio-sensor proteins by botulinumneurotoxin can be monitored by emission spectra scan in real time invitro. A): BoNT/B were pre-reduced with 2 mM DTT, 10 μM ZnCl₂ for 30 minat 37° C. 50 nM toxin were added into a cuvette that contained 300 nMCFP-SybII-YFP protein in the Hepes buffer (50 mM Hepes, 2 mM DTT, 10 μMZnCl₂). The emission spectra was recorded as described in FIG. 2A atindicated time before and after adding toxin (upper panel). 30 μlsamples were taken from the cuvette after each emission scan, and mixedwith SDS-loading buffer. These samples were subject to SDS-page andenhanced chemilluminescence (ECL). The cleavage of CFP-SybII-YFP fusionprotein was detected using an anti-his₆ antibody that recognizes thehis₆ tag at the fusion protein N-terminus (lower panel). The cleavage ofCFP-SybII-YFP fusion protein resulted in decreased YFP fluorescence andincreased CFP fluorescence. This change was recorded in real-time byemission spectra scan. B): CFP-SybII-YFP was used to test BoNT/Factivity, as described in panel A. C): CFP-SNAP-25-YFP was used to testBoNT/A activity (10 nM toxin was used), as described in panel A. D):CFP-SNAP-25-YFP was used to test BoNT/E activity (10 nM toxin was used),as described in panel A.

FIG. 4 shows the monitoring of botulinum neurotoxin protease kineticsusing bio-sensor proteins in a microplate spectrofluorometer. A):Fluorescence change during the cleavage of bio-sensor proteins bybotulinum neurotoxin could be recorded in real time using aplate-reader. 10 nM BoNT/A were mixed with 300 nM CFP-SNAP-25-YFP, and100 μl per well sample was scanned using a plater-reader. The excitationis 434 nm, and for each data point, both emission value at 470 nm (CFPchannel), and 527 nm (YFP or FRET channel) were collected. The reactionwas traced for one and half hour at the interval of 30 sec per datapoint. The decrease of YFP fluorescence and the increase of CFPfluorescence were monitored in real time. B): The rate of cleavage isdependent on the concentration of the neurotoxin. The variousconcentrations of botulinum neurotoxin A and E were tested for theirability to cleave the same amount of bio-sensor proteins. FRET signalchange (FRET ratio) is measured by the ratio between YFP emission signaland the CFP emission signal at the same data point. C): CFP-SNAP-25-YFPprotein alone, and the CFP/YFP protein mixture (1:1) were scanned at thesame time, as the internal control.

FIG. 5 shows the sensitivity of the bio-sensor assay using aplate-reader. A): 300 nM CFP-SNAP-25-YFP were mixed with variousconcentration of BoNT/A or E in a 96-well plate, the total volume is 100μl per well. The plate was incubated at 37° C. for 4 hours and thenscanned with a plate-reader (upper panel). The FRET ratio was plottedagainst the log value of the toxin concentration. The EC₅₀ values foreach curve are listed in the table on the lower panel. Each data pointrepresents the mean of three independent experiments. B): 300 nMCFP-SybII-YFP were mixed with various concentration of BoNT/B or F. Thedata were collected and plotted as described in panel A.

FIG. 6 depicts the monitoring of botulinum neurotoxin activity in livingcells. A): CFP-SNAP-25-YFP was expressed in wild type PC12 cells. Theentry and catalytic activity of BoNT/A (50 nM) was monitored byrecording the FRET ratio change that results from CFP-SNAP-25-YFPcleavage inside the cells. The FRET ratio was averaged from a total of53 toxin treated cells and 53 control cells. Treatment with BoNT/A for72 hours reduced the FRET ratio of the entire population of cells by asignificant degree (P<1.47E-5). B): PC 12 cells that express syt II weretransfected with CFP-SybII-YFP and treated with BoNT/B (30 nM). Theentry and catalytic activity of BoNT/B were monitored by recording theFRET ratio change as in panel (A); 73 toxin treated and 73 control cellswere analyzed. Treatment with BoNT/B for 72 hours reduced the FRET ratioof the entire population of cells by a significant degree (P<2E-10).

FIG. 7 shows the monitoring BoNT/A activity in living cells usingaccording to the present invention. (a). Measuring the FRET signal oftoxin sensors in living cells. CFP-SNAP-25(141-206)-YFP was used totransfect PC12 cells. This sensor appeared to be soluble in cells. Threeimages using different filter set (CFP, FRET and YFP) were taken foreach cell sequentially, using exactly the same settings. Images werecolor coded to reflect the fluorescence intensity in arbitrary units asindicated in the look-up table on the left. The corrected FRET value wascalculated by subtracting the cross-talk from both CFP and YFP from thesignals collected using the FRET filter set, as detailed in the Methods.(b). PC12 cells transfected with CFP-SNAP-25(141-206)-YFP were used todetect BoNT/A activity. Fifty nM BoNT/A holotoxin was added to theculture medium and 80 cells were analyzed after 96 hours. The correctedFRET signal was normalized to the CFP fluorescence signal and plotted asa histogram with the indicated bins. Control cells were transfected withthe same sensor but were not treated with toxins, and they were analyzedin parallel. Incubation with BoNT/A shifted the FRET ratio (correctedFRET/CFP) among the cell population, indicating the sensor proteins werecleaved by BoNT/A in cells. However, the shift was small, indicatingthat the cleavage was not efficient in cells. (c). Left panel: anefficient toxin sensor was built by linking CFP and YFP throughfull-length SNAP-25 (amino acid 1-206), and tested for detecting BoNT/Aactivity in cells. This CFP-SNAP-25(FL)-YFP fusion protein was localizedprimarily to plasma membranes in cells via palmitoylation at its fourcysteines (left panel, upper frames of the middle panel). Middle panel:PC12 cells were transfected with the CFP-SNAP-25(FL)-YFP sensor and usedto detect BoNT/A activity. Fifty nM BoNT/A holotoxin was added to theculture medium and the FRET signals of 200 cells were analyzed after 48and 96 hours as described in panel (a). Control cells were transfectedwith toxin sensors but were not treated with toxins, and they wereanalyzed in parallel. The images of representative cells were shown inthe middle panel. This sensor yielded significant FRET (upper “correctedFRET” frame of the middle panel). The FRET signal was abolished aftercells were treated with BoNT/A (96 h, lower “corrected FRET” frame ofthe middle panel). Note: one of the cleavage products, the C-terminus ofSNAP-25 tagged with YFP, was degraded after toxin cleavage. Thus, thefluorescence signal of YFP was significantly decreased in toxin-treatedcells (lower “YFP” frame). Right panel: the FRET ratios are plotted as ahistogram with indicated bins as described in panel (b). (d). PC12 cellswere transfected with CFP-SNAP-25(Cys-Ala)-YFP (full length SNAP-25 withCys 85,88,90,92 Ala mutations, left panel). This protein has diffuselydistributed throughout the cytosol, and lacked the strong FRET signalobserved for CFP-SNAP-25(FL)-YFP (right panel, “corrected FRET” frame).(e). PC12 cells were transfected with CFP-SNAP-25(FL)-YFP andCFP-SNAP-25(Cys-Ala)-YFP. Cells were then treated with (+, intact cells)or without (−, intact cells) BoNT/A (50 nM, 72 h), and were harvested.Half of the cell extracts from samples that are not been exposed toBoNT/A were also incubated with (+, in vitro) or without (−, in vitro)reduced BoNT/A in vitro (200 nM, 30 min, 37° C.), served as controls toshow the cleavage products (two cleavage products are indicated byarrows). The same amount of each sample (30 μg cell lysate) was loadedto one SDS-page gel and subjected to immunoblot analysis using ananti-GFP antibody. While CFP-SNAP-25(FL)-YFP underwent significantcleavage in intact cells, there was no detectable cleavage ofCFP-SNAP-25(Cys-Ala)-YFP in cells, indicating the membrane anchoring isimportant for efficient cleavage by BoNT/A in living cells. Note: onlyone cleavage product (CFP-SNAP-25(1-197)) was detected in toxin treatedcells, indicating that the other cleavage product (SNAP-25(198-206)-YFP)was largely degraded in cells.

FIG. 8 shows that anchoring CFP-SNAP-25(141-206)-YFP sensor to theplasma membrane created a sensor that was efficiently cleaved by BoNT/Ain cells. (a). A schematic description of the construct built to targetCFP-SNAP-25(141-206)-YFP to the plasma membrane. A fragment of SNAP-25that contains the palmitoylation sites (residues 83-120) (SEQ ID NO: 11)was fused to the N-terminus of the CFP-SNAP-25(141-206)-YFP sensor, andthis fragment targeted the fusion protein to the plasma membrane. (b).PC12 cells were transfected withSNAP-25(83-120)-CFP-SNAP-25(141-206)-YFP. Fifty nM BoNT/A holotoxin wasadded to the culture medium and the FRET signals of 80 cells wereanalyzed after 96 hours as described in FIG. 7 a. Control cells,transfected with toxin sensors but not treated with toxins, wereanalyzed in parallel. The images of representative cells are shown inthe left panel. This sensor yielded significant FRET (upper “correctedFRET” frame of the left panel). The FRET signal was reduced after cellswere treated with BoNT/A (96 h, lower “corrected FRET” frame of the leftpanel). Right panel: the FRET ratios of cells are plotted as a histogramwith indicated bins as described in FIG. 7 b. (c). PC12 cells weretransfected with various CFP/YFP constructs and the corresponding FRETratios were determined as described in FIG. 7 a. Co-expression of CFPand YFP in cells, did not result in significant FRET under our assayconditions. CFP-SNAP-25(FL)-YFP exhibited significant levels of FRETwhereas the soluble CFP-SNAP-25(Cys-Ala)-YFP did not.

FIG. 9 shows that efficient cleavage of Syb by BoNT/B requires thelocalization of Syb to vesicles. (a). CFP-Syb(33-94)-YFP was used totransfect a PC12 cell line that stably expresses synaptotagmin II (Donget al. Synaptotagmins I and II mediate entry of botulinum neurotoxin Binto cells. J. Cell Biol. 162, 1293-1303 (2003)). This sensor appears tobe soluble inside cells and generates strong FRET signals (upper panel).PC12 cells transfected with CFP-Syb(33-94)-YFP were used to detectBoNT/B activity. Fifty nM BoNT/B holotoxin was added to the culturemedium and 80 cells were analyzed after 96 hours as described in FIG. 7b. Control cells were transfected with the same sensor but were nottreated with toxins, and they were analyzed in parallel. Incubation withBoNT/B shifted the FRET ratio among the cell population, indicating thesensor proteins were cleaved by BoNT/B in cells. However, the shift wassmall, indicating that the cleavage was not efficient in cells. (b). Aschematic description of YFP-Syb(FL)-CFP sensor. Full-length Syb contain116 amino acids, and is localized to vesicles through a singletransmembrane domain. Cleavage of Syb by BoNT/B released the cytoplasmicdomain of Syb tagged with YFP from the vesicle. (c). PC12 cells thatstably express synaptotagmin II were transfected with YFP-Syb(FL)-CFP,and were treated with BoNT/B (50 nM, 48 h, lower frames), or withouttoxin (control, upper frames). CFP and YFP fluorescence images werecollected for each cell, and representative cells are shown. This sensoris localized to vesicles, and was excluded from the nucleus in livingcells, as evidenced by both CFP and YFP fluorescent signals (upperframes). BoNT/B treatment resulted in a redistribution of YFP signals,which became soluble in the cytosol and entered the nucleus. (d). Atruncated version of Syb, residues 33-116 (SEQ ID NO: 10), was used tolink a CFP and YFP. This construct contains the same cytosolic region(residues 33-94, panel (b)) as the Syb fragments in the soluble sensorCFP-Syb(33-96)-YFP, and it also contains the transmembrane domain ofSyb. PC12 cells that express synaptotagmin II were transfected withCFP-Syb(33-116)-YFP and CFP-Syb(33-94)-YFP. Cells were then treated with(+, intact cells) or without (−, intact cells) BoNT/B (50 nM, 48 h), andwere harvested. Half of the cell extracts from samples that were notexposed to BoNT/B were also incubated with (+, in vitro) or without (−,in vitro) reduced BoNT/B in vitro (200 nM, 30 min, 37.degree. C.). Twocleavage products are indicated by asterisks. The same amount of eachsample (30.mu.g cell lysate) was loaded to one SDS-page gel andsubjected to immunoblot analysis using an anti-GFP antibody. WhileCFP-Syb(33-116)-YFP underwent significant cleavage in intact cells,there was no detectable cleavage of CFP-Syb(33-94)-YFP, indicating thelocalization to vesicles is important for efficient cleavage by BoNT/Bin living cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel compositions and methods based onfluorescence resonance energy transfer (FRET) between fluorophoreslinked by a peptide linker which is a substrate of a BoNT and can becleaved by the toxin, to detect botulinum neurotoxins and monitor theirsubstrate cleavage activity, preferably in real time. The method andcompositions of the present invention allow for the detection ofpico-molar level BoNTs within hours, and can trace toxin enzymatickinetics in real time. The methods and compositions can further be usedin high-throughput assay systems for large-scale screening of toxininhibitors, including inhibitors of toxin cellular entry andtranslocation through vesicle membrane using cultured cells. The presentinvention is also suitable for monitoring botulinum neurotoxin activityin living cells and neurons.

In another embodiment, the present invention provides a construct andmethod of using the construct which comprises full-length SNAP-25 andSyb proteins as the linkers, as fluorescent biosensors that can detecttoxin activity within living cells. Cleavage of SNAP-25 abolishedCFP/YFP FRET signals and cleavage of Syb resulted in spatialredistribution of the YFP fluorescence in cells. The present inventionprovides a means to carry out cell based screening of toxin inhibitorsand for characterizing toxin activity inside cells. The presentinvention also discloses that the sub-cellular localization of SNAP-25and Syb affects efficient cleavage by BoNT/A and B in cells,respectively.

Fluorescent Resonance Energy Transfer (FRET) is a tool which allows theassessment of the distance between one molecule and another (e.g. aprotein or nucleic acid) or between two positions on the same molecule.FRET is now widely known in the art (for a review, see Matyus, (1992) J.Photochem. Photobiol. B: Biol., 12:323). FRET is a radiationless processin which energy is transferred from an excited donor molecule to anacceptor molecule. Radiationless energy transfer is thequantum-mechanical process by which the energy of the excited state ofone fluorophore is transferred without actual photon emission to asecond fluorophore. The quantum physical principles are reviewed inJovin and Jovin, 1989, Cell Structure and Function byMicrospectrofluorometry, eds. E. Kohen and J. G. Hirschberg, AcademicPress. Briefly, a fluorophore absorbs light energy at a characteristicwavelength. This wavelength is also known as the excitation wavelength.The energy absorbed by a fluorochrome is subsequently released throughvarious pathways, one being emission of photons to produce fluorescence.The wavelength of light being emitted is known as the emissionwavelength and is an inherent characteristic of a particularfluorophore. In FRET, that energy is released at the emission wavelengthof the second fluorophore. The first fluorophore is generally termed thedonor (D) and has an excited state of higher energy than that of thesecond fluorophore, termed the acceptor (A).

An essential feature of the process is that the emission spectrum of thedonor overlap with the excitation spectrum of the acceptor, and that thedonor and acceptor be sufficiently close.

In addition, the distance between D and A must be sufficiently small toallow the radiationless transfer of energy between the fluorophores.Because the rate of energy transfer is inversely proportional to thesixth power of the distance between the donor and acceptor, the energytransfer efficiency is extremely sensitive to distance changes. Energytransfer is said to occur with detectable efficiency in the 1-10 nmdistance range, but is typically 4-6 nm for optimal results. Thedistance range over which radiationless energy transfer is effectivedepends on many other factors as well, including the fluorescencequantum efficiency of the donor, the extinction coefficient of theacceptor, the degree of overlap of their respective spectra, therefractive index of the medium, and the relative orientation of thetransition moments of the two fluorophores.

The present invention provides a construct (“FRET construct”) whichcomprises a fluorophore FRET donor and an acceptor linked by linkerpeptide (“substrate peptide”) that is cleavable by a corresponding BoNT.In the presence of a BoNT, the linker peptide is cleaved, therebyleading to a decrease in energy transfer and increased emission of lightby the donor fluorophore. In this way, the proteolysis activity of thetoxin can be monitored and quantitated in real-time.

As used herein with respect to donor and corresponding acceptorfluorescent moieties, “corresponding” refers to an acceptor fluorescentmoiety having an emission spectrum that overlaps the excitation spectrumof the donor fluorescent moiety. The wavelength maximum of the emissionspectrum of the acceptor fluorescent moiety should be at least 100 nmgreater than the wavelength maximum of the excitation spectrum of thedonor fluorescent moiety. Accordingly, efficient non-radioactive energytransfer can be produced.

As used herein with respect to substrate peptide and BoNT,“corresponding” refers to a BoNT toxin that is capable of acting on thelinker peptide and cleaves at a specific cleavage site.

Fluorescent donor and corresponding acceptor moieties are generallychosen for (a) high efficiency Forster energy transfer; (b) a largefinal Stokes shift (>100 nm); (c) shift of the emission as far aspossible into the red portion of the visible spectrum (>600 nm); and (d)shift of the emission to a higher wavelength than the Raman waterfluorescent emission produced by excitation at the donor excitationwavelength. For example, a donor fluorescent moiety can be chosen thathas its excitation maximum near a laser line (for example,Helium-Cadmium 442 nm or Argon 488 nm), a high extinction coefficient, ahigh quantum yield, and a good overlap of its fluorescent emission withthe excitation spectrum of the corresponding acceptor fluorescentmoiety. A corresponding acceptor fluorescent moiety can be chosen thathas a high extinction coefficient, a high quantum yield, a good overlapof its excitation with the emission of the donor fluorescent moiety, andemission in the red part of the visible spectrum (>600 nm).

A skilled artisan will recognize that many fluorophore molecules aresuitable for FRET. In a preferred embodiment, fluorescent proteins areused as fluorophores. Representative donor fluorescent moieties that canbe used with various acceptor fluorescent moieties in FRET technologyinclude fluorescein, Lucifer Yellow, B-phycoerythrin,9-acridineisothiocyanate, Lucifer Yellow VS,4-acetamido-4′-isothio-cyanatostilbene-2,2′-disulfonic acid,7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl1-pyrenebutyrate, and4-acetamido-4′-isothiocyanatostilbene-2-,2′-disulfonic acid derivatives.Representative acceptor fluorescent moieties, depending upon the donorfluorescent moiety used, include LC-Red 640, LC-Red 705, Cy5, Cy5.5,Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamineisothiocyanate, rhodamine×isothiocyanate, erythrosine isothiocyanate,fluorescein, diethylenetriamine pentaacetate or other chelates ofLanthanide ions (e.g., Europium, or Terbium). Donor and acceptorfluorescent moieties can be obtained, for example, from Molecular Probes(Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

Table 1 lists others examples of chemical fluorophores suitable for usein the invention, along with their excitation and emission wavelengths.

Certain naturally occurring amino acids, such as tryptophan, arefluorescent. Amino acids may also be derivatized, e.g. by linking afluorescent group onto an amino acid (such as linking AEDANS to a Cys),to create a fluorophore pair for FRET. The AEDANS-Cys pair is commonlyused to detect protein conformational change and interactions. Someother forms fluorescence groups have also been used to modify aminoacids and to generate FRET within the protein fragments (e.g.2,4-dinitrophenyl-lysine withS—(N-[4-methyl-7-dimethylamino-coumarin-3-yl]-carboxamidomethyl)-cysteine).

In another embodiment, which is especially suitable for using in livecells, green fluorescent protein (GFP) and its various mutants are usedas the fluorophores. Examples of fluorescent proteins which vary amongthemselves in excitation and emission maxima are listed in Table 1 of WO97/28261 (Tsien et al., 1997), which is incorporated herein byreference. These (each followed by [excitation max./emission max.]wavelengths expressed in nanometers) include wild-type Green FluorescentProtein [395(475)/508] and the cloned mutant of Green FluorescentProtein variants P4 [383/447], P4-3 [381/445], W7 [433(453)/475(501)],W2 [432(453)/408], S65T [489/511], P4-1 [504(396)/480], S65A [471/504],S65C [479/507], S65L [484/510], Y66F [360/442], Y66W [458/480], I0c[513/527], W1B [432(453)/476(503)], Emerald [487/508] and Sapphire[395/511]. Red fluorescent proteins such as DsRed (Clontech) having anexcitation maximum of 558 nm and an emission maximum of 583 can also beused. This list is not exhaustive of fluorescent proteins known in theart; additional examples are found in the Genbank and SwissPro publicdatabases.

TABLE 1 Fluorophore Excitation (nm) Emission (nm) Color PKH2 490 504green PKH67 490 502 green Fluorescein (FITC) 495 525 green Hoechst 33258360 470 blue R-Phycoerythrin (PE) 488 578 orange-red Rhodamine (TRITC)552 570 red Quantum Red ™ 488 670 red PKH26 551 567 red Texas Red 596620 red Cy3 552 570 red

GFP is a 27-30 KD protein, and can be fused with another protein, e.g.the target protein, and the fusion protein can be engineered to beexpressed in a host cell, such as those of E. coli. GFP and its variousmutants are capable of generating fluorescence in live cells andtissues. The mutation forms of GFP has slight amino acid differenceswithin their fluorescence region which result in shifted spectrum. Moremutations of GFP are expected to be created in the future to havedistinct spectra. Among these GFP variants, BFP-YFP, BFP-CFP, CFP-YFP,GFP-DsRed, are commonly used as FRET donor-acceptor pairs to detectprotein-protein interactions. These pairs are also suitable to detectprotease cleavage of the linker region that links the pair.

The use of fluorescent proteins is preferred because they enable the useof a linker fragment of about 60 amino acids residues. Longer fragmentsusually are more sensitive to toxin recognition and cleavage, thus,results in a higher sensitivity for detecting toxin. As shown in theexamples below, the EC50 for BoNT/A and E after 4 hour incubation withCFP-SNAP-YFP are as low as 15-20 pM (1-2 ng/ml), when measured with awidely used microplate spectrofluorometer (Spectra Max Gemini, MolecularDevice). EC50 for BoNT/B and F are about 200-250 pM, and the sensitivitycan be enhanced by increasing incubation time.

According to one embodiment of the present invention, two fluorophoresare linked together by a linker of suitable length, such that FREToccurs. The linker is a fragment of a BoNT substrate protein. Whenexposed to a BoNT capable of cleaving the linker fragment, the twofluorophores are separated and FRET is abolished. The present inventionprovides accordingly a method for detecting BoNT by detecting the changein FRET. SNARE proteins from many species are suitable as substrateproteins for BoNT toxins, because these proteins are known to beconserved at the amino acid level. Many of these BoNT substrate proteinsare known and available to be used or modified for use as a suitablelinker peptide for the present invention. Some of the substrate proteinsand their GenBank accession numbers are listed in Table 2. The completeamino acid sequences for human SNAP25-A (NP 003072) and for human syb II(AAH19608) are provided in the Sequence Listing as SEQ ID NO: 7 and SEQID NO: 8, respectively.

TABLE 2 GenBank Protein Origin Accession # syb I mouse NP-033522 syb 1ahuman NP_055046 syb I rat AAN85832 syb African frog AAB88137 sybelectric ray A32146 syb California sea hare P35589 syb Takifugu rubripesAAB94047 syb drosophila AAB28707 syb II mouse NP_033523 syb II Africanfrog P47193 Syb II rabbit AAN14408 syb II rat NP_036795 syb II humanAAH19608 syb 3 human AAP36821 SNAP25-1 Zebra fish AAC64289 SNAP25-Ahuman NP_003072 SNAP25a American frog AAO13788 SNAP25 mouse XP_130450SNAP25 rat NP_112253 SNAP25 goldfish I50480 SNAP25-b Zebra fishNP_571509 SNAP25b American frog AAO13789 SNAP25-3 human CAC34535

Each BoNT toxin is known to cleave a specific peptide bond between twospecific amino acids within the toxin cleavage site. Table 3 below liststhe amino acid pairs for each BoNT toxin. These pairs of amino acidsequence, however, are not sufficient for toxin recognition andcleavage. For example, BoNT/A cleaves SNAP-25 at Q(197)-R(198) of therat SNAP-25 sequence (GenBank accession No: NP_(—)112253), but notQ(15)-R(16). Generally, there is no conserved amino acid sequence as therecognition site; rather, the toxins are believed to recognize thetertiary, rather than the primary, structure of their target protein.Nevertheless, a very short fragment of the substrate protein issufficient for toxin recognition and cleavage, regardless of its speciesorigin, as long as they have the two amino acid residues at the toxincleavage site listed above in Table 3 below.

The linker protein or peptide can be as long as the full-length of theBoNT substrate protein. Preferably the linker is a shorter fragment ofthe substrate protein. A full-length substrate linker may be too longfor efficient FRET, and a shorter fragment is more effective and easierto produce than the full-length protein. On the other hand, as indicatedabove, the linker peptide should be above certain minimum length,because below such a minimum length, cleavage of the linker peptide bythe respective BoNTs becomes inefficient.

TABLE 3 Peptide Bonds Recognized and Cleaved by BoNT Toxins CleavagePutative Minimum Toxin Site Recognition Sequence BoNT/A Q-RGlu-Ala-Asn-Gln- (SEQ ID NO: 1) Arg-Ala-Thr-Lys BoNT/B Q-FGly-Ala-Ser-Gln- (SEQ ID NO: 2) Phe-Glu-Thr-Ser BoNT/C R-AAla-Asn-Gln-Arg- (SEQ ID NO: 3) (SNAP25) Ala-Thr-Lys-Met BoNT/C K-AAsp-Thr-Lys-Lys- (SEQ ID NO: 4) (Syntaxin) Ala-Val-Lys-Phe BoNT/D K-LBoNT/E R-I Gln-Ile-Asp-Arg- (SEQ ID NO: 5) Ile-Met-Glu-Lys BoNT/F Q-KGlu-Arg-Asp-Gln- (SEQ ID NO: 6) Lys-Leu-Ser-Glu BoNT/G A—A

Using syb II and BoNT/B as an example, Table 4 below illustrates therelationship between linker-peptide length and toxin cleavage rate. Thefull-length rat syb II protein (GenBank No: NP_(—)036795) has 116 aminoacids, of which amino acid 1-94 at the amino terminus is the cytoplasmicdomain and the rest is the transmembrane domain. As Table 1 makes clear,within certain limit, a shorter fragment is cleaved by the toxin at aslower rate (data from Foran et al., Biochemistry 33:15365, 1994).

As can be seen from Table 4, tetanus neurotoxin (TeNT) requires a longerfragment (33-94) for optimum cleavage than BoNT/B (55-94). A fragmentconsisting of 60-94 has been used in several studies including severalpeptide-based toxin assay methods (Schmidt et al., 2003, supra, andSchmidt et al., 2001, Analytical Biochemistry, 296: 130-137).

For BoNT/A, the 141-206 fragment of SNAP-25 (SEQ ID NO: 9) is requiredfor retaining most of the toxin sensitivity (Washbourne et al., 1997,FEBS Letters, 418:1). There are also other reports that a shorterpeptide, amino acids 187-203 of SNAP25, is sufficient to be cleaved byBoNT/A (, 2001). The minimum site for BoNT/A is:Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys (SEQ ID NO: 1). BoNT/A cleaves betweenGln-Arg.

TABLE 4 Relationship Between Syb II fragment Length and Cleavage Ratesyb II Fragment Relative cleavage rate by % Relative cleavage rateLength BoNT/B by TeNT full length 1-116 100 (%) 100 (%) 33-94 100 10045-94 121 1.1 55-94 105 0.4 65-94 7 0.3

Using full-length SNAP-25 as the linker sequence between CFP and YFPinside PC12 cells, preliminary results indicate that FRET signalsobtained are stronger than those obtained using a shorter fragment,enough to be detected using a conventional lab microscope. It isbelieved that in PC12 cells the rate of cleavage of full-length SNAP-25by BoNT/A is faster and more consistent from cell to cell than the shortfragment, likely due to the fact that full-length SNAP-25 is targetedonto plasma membrane, on to which the BoNT/A light chain may also betargeted and anchored.

For BoNT/B, a fragment as short as between residues 60-94 was found tobe as effective as a fragment between residues 33-94. Preferably, afragment between 33-94 is used for BoNT/B and TeNT. Both toxins cleavebetween Gln and Phe, and the minimum sequence for cleavage is believedto be: Gly-Ala-Ser-Gln-Phe-Glu-Thr-Ser (SEQ ID NO: 2). There areindications that BoNT/B light chain may be targeted and anchored onsynaptic vesicles, it may be desirable to also target, via signalsequences, a FRET construct of the present invention onto synapticvesicles to achieve increased cleavage efficient inside cells.

BoNT/C cleaves both SNAP25 and Syntaxin, and is believed to cleave at avery slow rate if the substrate is in solution. Native SNAP25 andSyntaxin that reside on the cell membrane are cleaved most efficientlyby BoNT/C. The minimum cleavage sequence for SNAP25 is:Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met (SEQ ID NO: 3), where cleavage occursbetween Arg-Ala; for Syntaxin, the minimum cleavage sequence isAsp-Thr-Lys-Lys-Ala-Val-Lys-Phe (SEQ ID NO: 4), and cleavage occurs atLys-Ala.

BoNT/E requires a minimum sequence of: Gln-Ile-Asp-Arg-Ile-Met-Glu-Lys(SEQ ID NO: 5), and cleaves between Arg-Ile.

BoNT/F cleaves Gln-Lys. Schmidt et al. (Analytical Biochemistry, 296:130-137 (2001)) reported that a 37-75 fragment of syb II retains most oftoxin sensitivity, and the minimum sequence is:Glu-Arg-Asp-Gln-Lys-Leu-Ser-Glu (SEQ ID NO: 6).

From the above discussion on the minimum cleavage sites and therelationship between FRET signal strength and linker length, and betweencleavage efficiency and linker length, a person skilled in the art caneasily choose suitable linker length to achieve optimal balance betweenFRET signal strength and cleavage efficiency.

Preferably, the linker length is anywhere between about 8 a.a. to about100 a.a., preferably between 10-90, more preferable between 20-80,between 30-70, between 40-60 a.a. long, depending on the specificsubstrate and toxin combination.

In one embodiment, a linker protein or fragments thereof may be firstpurified, or peptides were first synthesized, and then the fluorescencegroups were added onto certain amino acids through chemical reaction. Afluorescent label is either attached to the linker polypeptide or,alternatively, a fluorescent protein is fused in-frame with a linkerpolypeptide, as described below. The above discussion makes clear thatwhile short substrate fragments are desirable for toxin detectionspecificity, longer fragments may be desirable for improved signalstrength or cleavage efficiency. It is readily recognized that when thesubstrate protein contains more than one recognition site for one BoNT,a position result alone will not be sufficient to identify whichspecific toxin is present in the sample. In one embodiment of thepresent invention, if a longer substrate fragment, especially afull-length substrate protein, is used, the substrate may be engineered,e.g. via site-directed mutagenesis or other molecular engineeringmethods well-known to those skilled in the art, such that it containsonly one toxin/protease recognition site. See e.g. Zhang et al, 2002,Neuron 34:599-611 “Ca2+-dependent synaptotagmin binding to SNAP-25 isessential for Ca2+ triggered exocytosis” (showing that SNAP-25 havingmutations at BoNT/E cleavage site (Asp 179 to Lys) is resistant toBoNT/E cleavage, but behaves normally when tested for SNARE complexformation). In a preferred embodiment, the method of the presentinvention uses a combination of specificity engineering and lengthoptimization to achieve optimal signal strength, cleavage efficiency andtoxin/serotype specificity.

In a preferred embodiment, the fluorophores are suitable fluorescentproteins linked by a suitable substrate peptide. A FRET construct maythen be produced via the expression of recombinant nucleic acidmolecules comprising an in-frame fusion of sequences encoding such apolypeptide and a fluorescent protein label either in vitro (e.g., usinga cell-free transcription/translation system, or instead using culturedcells transformed or transfected using methods well known in the art).Suitable cells for producing the FRET construct may be a bacterial,fungal, plant, or an animal cell. The FRET construct may also beproduced in vivo, for example in a transgenic plant, or in a transgenicanimal including, but not limited to, insects, amphibians, and mammals.A recombinant nucleic acid molecule of use in the invention may beconstructed and expressed by molecular methods well known in the art,and may additionally comprise sequences including, but not limited to,those which encode a tag (e.g., a histidine tag) to enable easypurification, a linker, a secretion signal, a nuclear localizationsignal or other primary sequence signal capable of targeting theconstruct to a particular cellular location, if it is so desired.

As low as 300 nM proteins is enough to generate sufficient fluorescencesignals that can be detected using a microplate spectrofluorometer. Thefluorescence signal change can be traced in real time to reflect thetoxin protease enzymatic activity. Real time monitoring measures signalchanges as a reaction progresses, and allows both rapid data collectionand yields information regarding reaction kinetics under variousconditions. FRET ratio changes and degrees of cleavage may becorrelated, for example for a certain spectrofluorometer using a methodsuch as HPLC assay in order to correlate the unit of kinetic constantfrom the FRET ratio to substrate concentration.

The method of the present invention is highly sensitive, and as aconsequence, can be used to detect trace amount of BoNTs inenvironmental samples directly, including protoxins inside Botulinumbacterial cells. Accordingly, the present invention further provides amethod for toxin detection and identification directly usingenvironmental samples.

The present invention further provides a method for screening forinhibitors of BoNTs using the above described in vitro system. Becauseof its high sensitivity, rapid readout, and ease of use An in vitrosystems based on the present invention is also suitable for screeningtoxin inhibitors. Specifically, a suitable BoNT substrate-FRET constructis exposed to a corresponding BoNT, in the presence of a candidateinhibitor substance, and changes in FRET signals are monitored todetermine whether the candidate inhibits the activities of the BoNT.

The present invention further provides for a method for detecting a BoNTusing a cell-based system for detecting BoNTs and further for screeningfor inhibitors of BoNTs. A suitable BoNT substrate-FRET construct asdescribed above is expressed inside a cell, and the cell is then exposedto a sample suspected of containing a BoNT, and changes in FRET signalsare then monitored as an indication of the presence/absence orconcentration of the BoNT. Specifically, a decrease in FRET signalsindicates that the sample contains a corresponding BoNT.

Cell-based high-throughput screening assays have the potential to revealnot only agents that can block proteolytic activity of the toxins, butalso agents that can block other steps in the action of the toxin suchas binding to its cellular receptor(s), light chain translocation acrossendosomal membranes and light chain refolding in the cytosol aftertranslocation.

The present invention further provides a method for screening forinhibitors of BoNTs using the above described cell-based system.Specifically, a cell expressing a suitable BoNT substrate-FRET constructis exposed to a corresponding BoNT, in the presence of a candidateinhibitor substance, and changes in FRET signals are monitored todetermine whether the candidate inhibits the activities of the BoNT.Compared to other in vitro based screening methods which can onlyidentify direct inhibitors of toxin-substrate interaction, thecell-based screening method of the present invention further allows forthe screening for inhibitors of other toxin-related activities, such asbut not limited to toxin-membrane receptor binding, membranetranslocation, and intra cellular toxin movement.

According to a preferred embodiment, a recombinant nucleic acidmolecule, preferably an expression vector, encoding a BoNT substratepolypeptide and two suitable FRET-effecting fluorescent peptides isintroduced into a suitable host cell. An ordinarily skilled person canchoose a suitable expression vector, preferably a mammalian expressionvector for the invention, and will recognize that there are enormousnumbers of choices. For example, the pcDNA series of vectors, such aspCI and pSi (from Promega, Madison, Wis.), CDM8, pCeo4. Many of thesevectors use viral promoters. Preferably, inducible promoters are used,such as the tet-off and tet-on vectors from BD Biosciences (San Jose,Calif.).

Many choices of cell lines are suitable as the host cell for the presentinvention. Preferably, the cell is of a type in which the respectiveBoNT exhibits its toxic activities. In other words, the cells preferablydisplays suitable cell surface receptors, or otherwise allow the toxinto be translocated into the cell sufficiently efficiently, and allow thetoxin to cleave the suitable substrate polypeptide. Specific examplesinclude primary cultured neurons (cortical neuron, hippocampal neuron,spinal cord motor neuron, etc); PC12 cells or derived PC12 cell lines;primary cultured chromaphin cells; several cultured neuroblastoma celllines, such as murine cholinergic Neuro 2a cell line, human adrenergicSK-N-SH cell line, and NS-26 cell line. See e.g. Foster and Stringer(1999), Genetic Regulatory Elements Introduced Into Neural Stem andProgenitor Cell Populations, Brain Pathology 9: 547-567.

The coding region for the substrate-FRET polypeptide is under thecontrol of a suitable promoter. Depending on the types of host cellsused, many suitable promoters are known and readily available in theart. Such promoters can be inducible or constitutive. A constitutivepromoter may be selected to direct the expression of the desiredpolypeptide of the present invention. Such an expression construct mayprovide additional advantages since it circumvents the need to culturethe expression hosts on a medium containing an inducing substrate.Examples of suitable promoters would be LTR, SV40 and CMV in mammaliansystems; E. coli lac or trp in bacterial systems; baculovirus polyhedronpromoter (polh) in insect systems and other promoters that are known tocontrol expression in eukaryotic and prokaryotic cells or their viruses.Examples of strong constitutive and/or inducible promoters which arepreferred for use in fungal expression hosts are those which areobtainable from the fungal genes for xylanase (xlnA), phytase,ATP-synthetase, subunit 9 (oliC), triose phosphate isomerase (tpi),alcohol dehydrogenase (AdhA), α-amylase (amy), amyloglucosidase (AG—fromthe glaA gene), acetamidase (amdS) and glyceraldehyde-3-phosphatedehydrogenase (gpd) promoters. Examples of strong yeast promoters arethose obtainable from the genes for alcohol dehydrogenase, lactase,3-phosphoglycerate kinase and triosephosphate isomerase. Examples ofstrong bacterial promoters include SP02 promoters as well as promotersfrom extracellular protease genes.

Hybrid promoters may also be used to improve inducible regulation of theexpression construct. The promoter can additionally include features toensure or to increase expression in a suitable host. For example, thefeatures can be conserved regions such as a Pribnow Box or a TATA box.The promoter may even contain other sequences to affect (such as tomaintain, enhance or decrease) the levels of expression of thenucleotide sequence of the present invention. For example, suitableother sequences include the Shl-intron or an ADH intron. Other sequencesinclude inducible elements—such as temperature, chemical, light orstress inducible elements. Also, suitable elements to enhancetranscription or translation may be present. An example of the latterelement is the TMV 5′ signal sequence (see Sleat, 1987, Gene 217:217-225; and Dawson, 1993, Plant Mol. Biol. 23: 97).

The expression vector may also contain sequences which act on thepromoter to amplify expression. For example, the SV40, CMV, and polyomacis-acting elements (enhancer) and a selectable marker can provide aphenotypic trait for selection (e.g. dihydrofolate reductase or neomycinresistance for mammalian cells or ampicillin/tetracyclin resistance forE. coli). Selection of the appropriate vector containing the appropriatepromoter and selection marker is well within the level of those skilledin the art.

Preferably the coding region for the substrate-FRET polypeptide is underthe control of an inducible promoter. In comparison to a constitutivepromoter, an inducible promoter is preferable because it allows forsuitable control of the concentration of the reporter in the cell,therefore the measurement of changes in FRET signals are greatlyfacilitated.

For example, FRET reporter can be controlled using the Tet-on & Tet-offsystem (BD Biosciences, San Jose, Calif.). Under the control of thispromoter, gene expression can be regulated in a precise, reversible andquantitative manner. Briefly, for Tet-on system, the transcription ofdownstream gene only happens when doxycycline is present in the culturemedium. After the transcription for a certain period of time, we canchange culture medium to deplete doxycycline, thus, stop the synthesisof new FRET reporter proteins. Therefore, there is no background fromnewly synthesized FRET proteins, and we may be able to see a fasterchange after toxin treatment.

Fluorescent analysis can be carried out using, for example, a photoncounting epifluorescent microscope system (containing the appropriatedichroic mirror and filters for monitoring fluorescent emission at theparticular range), a photon counting photomultiplier system or afluorometer. Excitation to initiate energy transfer can be carried outwith an argon ion laser, a high intensity mercury (Hg) arc lamp, a fiberoptic light source, or other high intensity light source appropriatelyfiltered for excitation in the desired range. It will be apparent tothose skilled in the art that excitation/detection means can beaugmented by the incorporation of photomultiplier means to enhancedetection sensitivity. For example, the two photon cross correlationmethod may be used to achieve the detection on a single-molecule scale(see e.g. Kohl et al., Proc. Nat'l. Acad. Sci., 99:12161, 2002).

A number of parameters of fluorescence output may be measured. Theyinclude: 1) measuring fluorescence emitted at the emission wavelength ofthe acceptor (A) and donor (D) and determining the extent of energytransfer by the ratio of their emission amplitudes; 2) measuring thefluorescence lifetime of D; 3) measuring the rate of photobleaching ofD; 4) measuring the anisotropy of D and/or A; or 5) measuring the Stokesshift monomer/excimer fluorescence. See e.g. Mochizuki et al., (2001)“Spatio-temporal images of grow-factor-induced activation of Ras andRap1.” Nature 411:1065-1068, Sato et al. (2002) “Fluorescent indicatorsfor imaging protein phosphorylation in single living cells.” NatBiotechnol. 20:287-294.

In another embodiment, the present invention provides a method fordetecting BoNTs using surface plasmon resonance imaging (SPRi)techniques. Surface plasmon resonance (SPR) is an established opticaltechnique for the detection of molecular binding, based on thegeneration of surface plasmons in a thin metal film (typically gold)that supports the binding chemistry. Surface plasmons are collectiveoscillations of free electrons constrained in the metal film. Theseelectrons are excited resonantly by a light field incident from a highlyrefractive index prism. The angle of incidence q over which thisresonant excitation occurs is relatively narrow, and is characterized bya reduction in the intensity of the reflected light which has a minimumat the resonant angle of incidence qr. The phase of the reflected lightalso varies nearly linearly with respect to q in this region. The valueof qr is sensitive to the refractive index of the medium that resideswithin a few nanometers of the metal film. Small variations in therefractive index, due to the binding of a molecule to the film, or dueto the change in the molecular weight of the bound molecules, maytherefore be detected as a variation of this angle. Many methods areknown in the art for anchoring biomolecules to metal surfaces, fordetecting such anchoring and measuring SPRi are known in the art, seee.g. U.S. Pat. No. 6,127,129 U.S. Pat. No. 6,330,062, and Lee et al.,2001, Anal. Chem. 73: 5527-5531, Brockman et al., 1999, J. Am. Chem.Soc. 121: 8044-8051, and Brockman et al., 2000, Annu. Rev. Phys. Chem.51: 41-63, which are all incorporated herein by reference in theirentirety.

In practice a layer of BoNT target peptides may be deposited on themetal. A sample suspected of containing a corresponding BoNT is appliedto the composite surface, and incubated to allow the toxin, if present,to cleave the bound target peptides. The cleavage will result in thedecrease of the molecular weight of the peptide bound to the metalsurface, which decrease can then be detected using standard equipmentsand methods. A decrease in the thickness of the bound layer indicatesthat cleavage has occurred and in consequence, the sample contains thetoxin corresponding to the target peptide.

Alternatively, binding of a BoNT protein to its corresponding substratepeptide, which is anchored to the metal surface, will also cause achange in the refractive index and can be detected by known SPRitechniques and apparati.

Many methods are known in the art for anchoring or depositing protein orpeptides molecules on to a metal surface. For example, add an extra Cysresidue can be added to the end of the peptide, which can then becrosslinked onto the metal surface. Indirectly, an antibody can beanchored first to the metal surface, and to which antibody the toxinsubstrate can be bound. Indirect anchoring via antibodies is suitablefor the present invention so long as the antibody-substrate binding doesnot prevent the toxin from recognizing and accessing the cleavage siteof the substrate. Furthermore, nickel-NTA or glutathione that can beused to hold down his6 or GST fusion proteins, respectively. Additionalinformation regarding anchoring peptide to metal surface may be found inWegner et al, (2002) Characterization and Optimization of Peptide Arraysfor the Study of pitope-Antibody Interactions Using Surface PlasmonResonance Imaging” Analytical Chemistry 74:5161-5168, which is alsoincorporated herein by reference in its entirety.

Changes of about 10-16 bases in a nucleic acid molecule, correspondingto 3,000 to 6,400 d in molecular weight, can be easily detected by SPRi.This implies that a change of as few as 16 amino acid residues in apeptide molecule can be detected. This high sensitivity allows theanchoring of a short peptide substrate onto the surface, instead ofusing the full-length toxin substrate proteins. Short peptide fragmentsare preferred because they are more stable, less expensive to prepareand allow higher reaction specificity.

EXAMPLES

Materials and Methods

Construction of bio-sensor DNA constructs: YFP cDNA (Clontech) wasinserted into the pECFP-C1 vector (Clontech) using EcoRI and BamHI siteto generate pECFP-YFP vector. cDNA encoding amino acid 33-94 of rat sybII was amplified using PCR and into pECFP-YFP vector using XhoI andEcoRI sites, which are between CFP and YFP gene, to generateCFP-SybII-YFP (also referred to as CFP-Syb (33-94)-YFP) construct thatcan be used to transfect cells. Construct CFP-SNAP-25-YFP (also referredto as CFP-SNAP-25 (141-206)-YFP) was built using the same method, butusing residues 141-206 of SNAP-25. A construct (CFP-SNAP25FL-YFP) withfull-length rat SNAP-25B as the linker was also made. In order to purifyrecombinant chimera proteins using bacteria E. coli, we also movedCFP-SybII-YFP gene and CFP-SNAP-25-YFP gene from pECFP-YFP vector into apTrc-his (Invitrogen) vector using NheI and BamHI sites.

The mutation of four Cys residues of SNAP-25 to Ala was accomplished bysite-directed mutagenesis using PCR, and the fragment was then insertedbetween CFP and YFP as described above.SNAP-25(83-120)-CFP-SNAP-25(141-206)-YFP were built by first insertingthe cDNA fragment that encoding the residues 83-120 of SNAP-25 into theXhoI/EcoRI sites of pEYFP-N1(Clontech), and then subcloningCFP-SNAP-25(141-206) cDNA into downstream sites using EcoRI/BamHI.YFP-Syb(FL)-CFP was built by first inserting a full length Syb II cDNAinto pECFP-C1 vectors at EcoRI and BamHI sites, and then inserting afull length YFP cDNA into the upstream at XhoI and EcoRI sites.YFP-Syb(33-116)-CFP was built by replacing full-length Syb inYFP-Syb(FL)-CFP construct via EcoRI/BamHI sites. All cDNA fragments weregenerated via PCR.

Protein purification and fluorescence spectra acquisition: His₆-taggedCFP-SybII-YFP and CFP-SNAP-25-YFP proteins were purified as described(Chapman et al., A novel function for the second C2 domain ofsynaptotagmin. Ca²⁺-triggered dimerization. J. Biol. Chem. 271,5844-5849 (1996)). Proteins were dialyzed using HEPES buffer (50 mMHEPES, pH 7.1) overnight. 300 nM protein was put into a cuvette in atotal volume of 500 μl HEPES buffer that contains 2 mM DTT and 10 μMZnCl₂. The emission spectra from 450 nM to 550 nM was collected using aPTIQM-1 fluorometer. The excitation wavelength is 434 nM, which is theoptimal excitation wavelength for CFP.

Activation of Botulinum neurotoxin and monitoring the cleavage ofbio-sensor proteins: BoNT/A, B, E or F was incubated with 2 mM DTT and10 μM ZnCl₂ for 30 min at 37° C. to reduce the toxin light chain fromthe heavy chain. For experiments using a PTIQM-1 fluorometer, 10 nMBoNT/A, E, or 50 nM BoNT/B, F were added into the cuvette that contains300 nM corresponding FRET sensors. The emission spectra were collectedas described above, at certain time intervals after adding toxins (e.g.0, 2, 5, 10, 30, 60, 90 min). At the end of each emission scan, a smallportion of the sample (30 μl) was collected, mixed with SDS-loadingbuffer, and later subjected onto a SDS-page gel. The sensor protein andthe cleavage products were visualized with an anti-his₆ antibody usingenhanced chemiluminescence (ECL) (Pierce).

For experiments using a spectrofluorometer, 300 nM FRET sensor proteinwere prepared in a 100 μl volume per well in a 96-well plate. Variousconcentrations of BoNTs were added into each well, and samples wereexcited at 434 nM. The emission spectra of YFP channel (527 nM), and CFPchannel (470 nM) were collected for 90 min at 30 sec interval. The FRETratio is determined by the ratio between YFP channel and CFP channel foreach data point.

Measure the FRET ratio change in live cells after toxin treatment DNAconstructs pECFP-SNAP25-YFP were used to transfect PC12 cells usingelectroporation (Bio-Rad). Cells were passed 24 hrs. after thetransfection, and 50 nM BoNT/A were added into the culture medium. Afterincubation for 72 hours with toxin, the fluorescence images of cellsthat express FRET sensor were collected using a Nikon TE-300 microscope.Two images of each cell (CFP channel and FRET channel) were collectedusing the following filter set (Chroma Inc.): CFP channel: CFPexcitation filter (436/10 nm), JP4 beamsplitter, CFP emission filter:(470/30 nm); FRET channel: CFP excitation filter (436/10 nm), JP4beamsplitter, YFP emission filter (535/30 nm). The background (the areasthat contain no cells) was subtracted from each image, and thefluorescence intensities of CFP channel and FRET channel of each cellwere compared using MetaMorph software. The FRET ratio is determined bythe intensity ratio between FRET channel and CFP channel as previousdescribed. Control cells were not treated with toxins but were analyzedin an identical manner. To test BoNT/B in live cells, we transfected aPC12 cell line that express syt II using the same procedure as describedabove.

Live cell imaging and FRET analysis: PC12 cells were transfected withvarious cDNA constructs indicated in the Figure legends viaelectroporation (Bio-Rad, CA). The fluorescence images were collectedusing a Nikon TE-300 microscope with a 100× oil-immersed objective.CFP/YFP FRET in live cells was quantified using an established methodwith the three-filter set method (Gordon et al., Quantitativefluorescence resonance energy transfer measurements using fluorescencemicroscopy. Biophys J. 74, 2702-2713 (1998); Sorkin et al., Interactionof EGF receptor and grb2 in living cells visualized by fluorescenceresonance energy transfer (FRET) microscopy. Curr. Biol. 10, 1395-1398(2000)). In brief, three consecutive images were acquired for each cell,through three different filter sets: CFP filter (excitation, 436/10 nm;emission, 470/30 nm), FRET filter (excitation, 436/10 nm; emission,535/30 nm), and YFP filter (excitation, 500/20 nm, emission, 535/30 nm).A JP4 beam splitter (Set ID 86000, Chroma Inc. VT) was used. All imageswere acquired with exact the same settings (4×4 Binning, 200 ms exposuretime). In order to exclude the concentration-dependent FRET signal thatcan arise from high expression level of fluorescence proteins, onlycells with CFP and YFP intensities below the half value of the maximal12-bit scale (1-2097 gray scale) were counted in our experiments(Miyawaki et al., Monitoring protein conformations and interactions byfluorescence resonance energy transfer between mutants of greenfluorescent protein. Methods Enzymol. 327, 472-500 (2000); Erickson etal., DsRed as a potential FRET partner with CFP and GFP. Biophys J 85,599-611 (2003)). The background (from areas that did not contain cells)was subtracted from each raw image before FRET values were calculated.The fluorescence intensity values of each image were then obtained andcompared. PC12 cells transfected with CFP or YFP alone were first testedin order to obtain the crosstalk value for these filter sets. The FRETfilter channel exhibits about 56-64% of bleed-through for CFP, and about24% for YFP. There is virtually no crosstalk for YFP while using the CFPfilter, or for CFP while using the YFP filter, which greatly simplifiedthe FRET calculations. For cells expressing toxin sensors, the“corrected FRET” value was calculated using the following equation:corrected FRET=FRET−(CFP×0.60)−(YFP×0.24), where FRET, CFP and YFPcorrespond to fluorescence intensity of images acquired through FRET,CFP and YFP filter sets, respectively. The average fraction ofbleed-through coming from CFP and YFP fluorescence are 0.6 and 0.24,respectively, when acquiring image through the FRET filter set. Becausetoxin cleavage of the CFP-SNAP25FL-YFP sensor resulted in the membranedissociation of YFP fragment, which was degraded in the cytosol (FIG. 7c, e), the FRET ratio used in our data analysis is calculated asnormalizing “corrected FRET” value to only the CFP fluorescenceintensity (corrected FRET/CFP). We note that the CFP intensity in thesecalculations was an underestimate due to donor quenching if FREToccurred. However, it has been reported the decrease in CFP fluorescencebecause of donor quenching is only about 5-10% (Gordon et al.,Quantitative fluorescence resonance energy transfer measurements usingfluorescence microscopy. Biophys J 74, 2702-2713 (1998); Sorkina et al.,Oligomerization of dopamine transporters visualized in living cells byfluorescence resonance energy transfer microscopy. J. Biol. Chem. 278,28274-28283 (2003)). All images and calculations were performed usingMetaMorph software (Universal Imaging Corp., PA).

For experiments involving toxin treatment, indicated holotoxins wereadded to the cell culture media for various time, and cells were thenanalyzed as described above. Control cells were transfected with toxinsensors but not treated with toxins, and they were analyzed in anidentical manner.

Immunoblot analysis of toxin substrate cleavage: Wild type PC12 cells orSyt II+PC12 cells (Dong et al., 2003, supra) were transfected withvarious toxin sensor cDNA constructs as indicated in the Figure legends.BoNT/A or B was added to the culture medium 24 h after transfection andcells were incubated for another 48 hrs. Cells were then harvested andcell lysates were subject to immunoblot analysis as describedpreviously. Control cells were transfected with the same cDNA constructsand assayed in parallel except they were not treated with toxins. Onethird of the control cell lysates were treated with toxins in vitro (200nM BoNT/A or B, 30 min at 37° C.), and subjected to immunoblot analysis.Endogenous SNAP-25 and transfected CFP-SNAP-25-YFP sensors were assayedusing an anti-SNAP-25 antibody 26. CFP-SNAP-25-YFP and CFP-SybII-YFPsensor proteins were also assayed using a GFP polyclonal antibody (SantaCruz, Calif.). An anti-his6 antibody (Qigen Inc., CA) was used to assayfor recombinant sensor protein cleavage.

Example 1 Bio-Sensors Based on CFP-YFP FRET Pair and BotulinumNeurotoxin Protease Activity

In order to monitor botulinum neurotoxin protease activity using FRETmethod, CFP and YFP protein are connected via syb II or SNAP-25fragment, denoted as CFP-SybII-YFP and CFP-SNAP-25-YFP, respectively(FIG. 1A). Short fragments of toxin substrates were used instead of thefull-length protein to optimize the CFP-YFP energy transfer efficiency,which falls exponentially as the distance increases. However, thecleavage efficiency by BoNTs decreases significantly as the targetprotein fragments get too short. Therefore, the region that containamino acid 33-96 of synaptobrevin sequence was used because it has beenreported to retain the same cleavage rate by BoNT/B, F, and TeNT as thefull length synaptobrevin protein does. Similarly, residues 141-206 ofSNAP-25 were selected to ensure that the construct can still berecognized and cleaved by BoNT/A and E.

The FRET assay is depicted in FIG. 1B. When excited at 434 nM (optimalexcitation wavelength for CFP), the CFP-SybII-YFP and CFP-SNAP-25-YFPchimera protein would elicit YFP fluorescence emission because of theFRET between CFP-YFP pair. Botulinum neurotoxins can recognize andcleave the short substrate fragments between CFP and YFP, and FRETsignal will be abolished after CFP and YFP are separated. Because thesechimera proteins can be expressed in living cells, they are also denotedas “bio-sensor” for botulinum neurotoxins.

We first purified his₆-tagged recombinant chimera protein ofCFP-SybII-YFP, and CFP-SNAP-25-YFP, and characterized their emissionspectra using a PTIQM-1 fluorometer. As expected, both bio-sensorproteins show an obvious YFP fluorescence peak at 525 nM when their CFPwere excited at 434 nM (FIG. 2B, C). On the contrary, the YFP alone onlygave small fluorescence signal when excited directly at 434 nM (FIG.2A). The mixture of individual CFP and YFP doesn't have the peakemission at 525 nM (FIG. 2A). This demonstrated the YFP fluorescencepeak observed using bio-sensor proteins resulted from FRET. Because theFRET ratio (YFP fluorescence intensity/CFP fluorescence intensity) wasaffected by many factors, such as buffer composition, the Zn²⁺concentration and the concentration of reducing agents (data not shown),the experiments thereafter were all carried out in the same bufferconditions (50 mM Hepes, 2 mM DTT, 10 μM ZnCl₂, pH 7.1). 2 mM DTT and 10μM Zn²⁺ were added to optimize the botulinum neurotoxin proteaseactivity.

Example 2 Monitoring the Cleavage of Bio-Sensor Proteins by BotulinumNeurotoxins In Vitro

300 nM chimera protein CFP-SybII-YFP was mixed with 50 nM pre-reducedBoNT/B holotoxin in a cuvette. The emission spectra were collected atdifferent time points after adding BoNT/B (0, 2, 5, 10, 30, 60 min,etc). At the end of each scan, a small volume of sample (30 μl) wastaken out from the cuvette and mixed with SDS-loading buffer. Thesesamples later were subjected to SDS-page gels and the cleavage ofchimera proteins were visualized using an antibody against the his₆ tagin the recombinant chimera protein. As shown in FIG. 3A, the incubationof bio-sensor protein with BoNT/B resulted in a decrease of YFP emissionand increase of CFP emission. The decrease of FRET ratio is consistentwith the degree of cleavage of the chimera protein by BoNT/B (FIG. 3A,low panel). This result demonstrates the cleavage of the bio-sensorprotein can be monitored in real time by recording the change in itsFRET ratio.

The same assay was performed to detect CFP-SybII-YFP cleavage by BoNT/F,and CFP-SNAP-25-YFP cleavage by BoNT/A or E (FIG. 3B, C, D). Similarresults were obtained with the experiment using BoNT/B. IN all cases, weobserved the same kinetics of cleavage of the substrate using both theoptical readout and the immunoblot blot analysis. BoNT/A and E cleavedtheir chimera substrate much faster than BoNT/B and F did in our assay.Thus, only 10 nM BoNT/A or E were used in order to record the changeoccurred within first several minutes. The cleavage of chimera proteinis specific, since mixing BoNT/B and F with CFP-SNAP-25-YFP, or mixingBoNT/A and E with CFP-SybII-YFP did not result in any change in FRETratio or substrate cleavage (data not shown).

Example 3 Monitoring Botulinum Neurotoxin Protease Activity in Real TimeUsing a Microplate Spectrofluorometer

The above experiments demonstrated that the activity of botulinumneurotoxin can be detected in vitro by monitoring the changes of theemission spectra of their target bio-sensor proteins. We then determinedif we could monitor the cleavage of bio-sensor proteins in real timeusing a microplate reader—this will demonstrate the feasibility to adaptthis assay for future high-throughput screening. As shown in FIG. 4A,300 nM CFP-SNAP-25-YFP chimera protein was mixed with 10 nM BoNT/A in a96-well plate. CFP was excited at 436 nm and the fluorescence of the CFPchannel (470 nM) and YFP channel (527 nM) were recorded over 90 min at30 sec intervals. Addition of BoNT/A resulted in the decrease of YFPchannel emission and the increase of CFP channel emission. This resultenabled us to trace the kinetics of botulinum neurotoxin enzymaticactivity in multiple samples in real time. For instance, as shown inFIG. 4B, various concentration of BoNT/A or E were added into 300 nMCFP-SNAP-25-YFP, and the FRET ration of each sample were monitoredsimultaneously as described in FIG. 4A. Changes in the FRET ratio wererelated to the toxin concentration—higher toxin concentration resultedin faster decrease of the FRET ratio. This change in FRET ratio isspecific, because no significant change was detected for eitherCFP-SNAP-25-YFP alone (FIG. 4C left panel) or a mixture of CFP and YFP(FIG. 4C right panel).

Although it would be difficult to correlate the FRET ratio change withthe actual cleavage of the bio-sensor proteins at this stage, thismethod still provides the easiest way to compare toxin cleavage kineticsamong multiple samples when these samples were prepared and scannedsimultaneously—it is particularly useful for high throughput screeningtoxin inhibitors because it would provide information about how theinhibitor affects toxin enzymatic activities. We note that the unit foreach kinetic parameter would be the FRET ratio instead of substratesconcentration in these cases.

The sensitivity of this FRET based assay is determined by incubatingvarious concentrations of toxins with fixed amount of their targetbio-sensor proteins for certain period of time. The FRET ratio isrecorded using a microplate spectro-fluorometer, and plotted againsttoxin concentration. As shown in FIG. 5A, this method has similarsensitivities for BoNT/A and E after 4 hours incubation (EC50 for BoNT/Ais 15 pM, and for BoNT/E is 20 pM, upper panel), and incubation for 16hours slightly increased the detection sensitivity (FIG. 5A, lowerpanel). The sensitivities for BoNT/B and F are close to each other, butare about 10 times lower than BoNT/A and E with 4 hours incubation (FIG.5B, upper panel, EC50 is 242 pM for BoNT/B, and 207 pM for BoNT/F).Extension of the incubation period to 16 hours increased the ability todetect BoNT/B and BoNT/F activity by 8-fold and 2-fold, respectively.

Example 4 Monitoring Botulinum Neurotoxin Activity in Live Cells

CFP-YFP based bio-sensor assay not only can be used to detect botulinumneurotoxin in vitro, but also can be used in live cells. To establishthis application, PC 12 cells were transfected with CFP-SNAP-25-YFP.PC12 cell is a neuroendocrine cell line that is able to take up BoNT/Aand E. Transfected cells were incubated with BoNT/A (50 nM) for 72hours, and the FRET ratio of cells that express CFP-SNAP25-YFP wererecorded using a epi-fluorescence microscope equipped with specialfilter sets for CFP-YFP FRET detection. Briefly, the FRET ratio iscalculated as the ratio between the fluorescence intensity of the imagesfrom the same cell collected using two filter sets, one for CFP(excitation 437 nm/emission 470 nm), and another for FRET (excitation437 nm/emission 535 nm). A total number of 53 cells were collected, andcompared to the same number of control cells which express the samebio-sensor protein but were not exposed to toxin. As shown in FIG. 6A,BoNT/A treatment for 72 hours significantly decreased FRET ratio for thecell population that was examined (p<1.47E-05). Wild type PC12 cells arenot sensitive to BoNT/B and F.

A PC12 cell line was recently created that expresses both synaptotagminII, a receptor for BoNT/B, and CFP-SybII-YFP bio-sensor. These cellswere used to detect BoNT/B action in live cells. As shown in FIG. 6B,BoNT/B (30 nM) treatment for 72 hours significantly decreased FRET ratioof the bio-sensor proteins expressed in cells (p<2.1E-10). We note thatthere were still large number of cells that do not appear to change FRETratio for both bio-sensor proteins. There are several possibleexplanations. First, the toxin/bio-sensor protein ratio may be too lowin these cells, thus, the significant cleavage of bio-sensor proteinsmay require a longer incubation time. Second, these cells may have highlevel of protein synthesis activity, thus new bio-sensor protein wassynthesis quickly to replace cleavage products. Nevertheless, theseexperiments demonstrate the feasibility to adopt this FRET based assayin living cells and neurons.

Example 5 Cell Based Detection of BoNTs

To carry out cell-based studies, we first transfected PC12 cells withCFP-SNAP-25(141-206)-YFP sensor (FIG. 7 a). The FRET signal in livingcells was acquired using an established three-filter set method with anepi-fluorescence microscope as shown in FIG. 2 a (Gordon, et al.,Quantitative fluorescence resonance energy transfer measurements usingfluorescence microscopy. Biophys. J. 74, 2702-2713 (1998); and Sorkin etal., Interaction of EGF receptor and grb2 in living cells visualized byfluorescence resonance energy transfer (FRET) microscopy. Curr. Biol.10, 1395-1398 (2000), as described above in the Materials and MethodsSection. Transfected PC12 cells were treated with 50 nM BoNT/A for 96hrs. Their fluorescence images were analyzed and the normalized FRETratio (corrected FRET/CFP) was plotted in FIG. 7 b. AlthoughSNAP-25(141-206) fragments were reported to have similar toxin cleavagerates as full length SNAP-25 in vitro (Washbourne et al., Botulinumneurotoxin types A and E require the SNARE motif in SNAP-25 forproteolysis. FEBS Lett. 418, 1-5 (1997)), CFP-SNAP-25(141-206)-YFPappeared to be a poor toxin substrate in living cells. Incubation ofBoNT/A (50 nM) for 96 hr exhibited a small (but significant) shift inthe FRET ratio among the cell population, indicating the cleavage isinefficient in cells, and this sensor is not practical for toxindetection in cells.

Surprisingly, we found that using full length SNAP-25 as the linkerbetween CFP and YFP yielded significant levels of FRET when expressed inPC12 cells, despite the fact that SNAP-25 is 206 amino acid residueslong (FIG. 7 c, and 8 c). This FRET signal is dependent on the membraneanchor of SNAP-25 since mutation of the palmitoylation sites withinSNAP-25 (Cys 85, 88, 90, 92 Ala) (Lane & Liu, Characterization of thepalmitoylation domain of SNAP-25. J. Neurochem. 69, 1864-1869 (1997);Gonzalo et al., SNAP-25 is targeted to the plasma membrane through anovel membrane-binding domain. J. Biol. Chem. 274, 21313-21318 (1999);Koticha et al., Plasma membrane targeting of SNAP-25 increases its localconcentration and is necessary for SNARE complex formation and regulatedexocytosis. J. Cell Sci. 115, 3341-3351 (2002); Gonelle-Gispert et al.,Membrane localization and biological activity of SNAP-25 cysteinemutants in insulin-secreting cells. J. Cell Sci. 113 (Pt 18), 3197-3205(2000)), which results in the cytosolic distribution of the protein(denoted as CFP-SNAP-25(Cys-Ala)-YFP), significantly reduced the FRETsignal (FIG. 7 d). This finding suggests the membrane anchoring ofSNAP-25 may result in conformational changes that bring N-terminus andC-terminus of SNAP-25 close to each other. This sensor is denoted asCFP-SNAP-25(FL)-YFP. Incubation of cells that expressCFP-SNAP-25(FL)-YFP with 50 nM BoNT/A resulted in a progressive decreasein the FRET ratio over time (FIG. 7 c, right panel). BoNT/A cleavage ofthe sensor resulted in two fragments: an N-terminal fragment of SNAP-25tagged with CFP that remained on the membrane (FIG. 2 c, middle panel),and a short C-terminal fragment of SNAP-25 tagged with YFP that wasexpected to redistribute into the cytosol after toxin cleavage.Interestingly, we noticed the C-terminal cleavage product,SNAP-25(198-206)-YFP, largely disappeared after the toxin cleavage (FIG.2 c, the “YFP” frame in the middle panel). This observation wasconfirmed by immunoblot analysis (FIG. 7 e), indicating this solublefragment was degraded much faster than the other fragment that isretained on the membrane. This unexpected result provides an alternativeway to detect toxin activity in living cells by simply monitoring theratio between CFP and YFP fluorescence.

It was recently reported that the BoNT/A light chain contains membranelocalization signals and targets to the plasma membrane indifferentiated PC12 cells (Fernandez-Salas et al., Plasma membranelocalization signals in the light chain of botulinum neurotoxin. Proc.Natl. Acad. Sci. USA 101, 3208-3213 (2004). Thus, we investigated thepossibility that membrane anchoring of SNAP-25 is critical for efficientcleavage by BoNT/A. To directly test this idea,CFP-SNAP-25(Cys-Ala)-YFP, which was distributed in the cytosol (FIG. 7d), and CFP-SNAP-25(FL)-YFP, which was anchored to the plasma membrane,were used to transfect PC12 cells and assayed in parallel for thecleavage by BoNT/A in cells. As indicated in FIG. 7 e, incubation ofcells with BoNT/A resulted in the cleavage of significant amount ofCFP-SNAP-25(FL)-YFP sensor, while there was no detectable cleavage ofCFP-SNAP-25 (Cys-Ala)-YFP under the same assay conditions.

To further confirm this finding, we also targeted the inefficient sensorthat contains SNAP-25(141-206) to the plasma membrane using a shortfragment of SNAP-25 (residues 83-120, which can target GFP to plasmamembranes (Gonzalo et al., SNAP-25 is targeted to the plasma membranethrough a novel membrane-binding domain. J. Biol. Chem. 274, 21313-21318(1999)) (FIG. 8 a). As expected, anchoring of theCFP-SNAP-25(141-206)-YFP sensor to the plasma membrane resulted inefficient cleavage by BoNT/A (FIG. 8 b). These findings indicate thatthe sub-cellular localization of SNAP-25 is indeed critical for theefficient cleavage by BoNT/A in cells.

We next tested whether the CFP-Syb(33-94)-YFP sensor could be used toassay BoNT/B activity in cells. For these studies, we used a PC12 cellline that expresses synaptotagmin II, which mediates BoNT/B entry intocells (Dong et al. Synaptotagmins I and II mediate entry of botulinumneurotoxin B into cells. J. Cell Biol. 162, 1293-1303 (2003)). As shownin FIG. 9 a(upper panel), transfected CFP-Syb(33-94)-YFP is a solubleprotein present throughout the cells. Similar to the case ofCFP-SNAP-25(141-206)-YFP, BoNT/B (50 nM, 96 hr) treatment only slightlydecreased the FRET ratio (FIG. 9 a, lower panel), indicating that thecleavage of this sensor is also inefficient in cells.

Our experience with the BoNT/A sensor prompted us to investigate whetherthe sub-cellular localization of Syb is important for cleavage of Syb byBoNT/B. Endogenous Syb is 116 amino acid residues long, and resides onsecretory vesicles through a single transmembrane domain (residues95-116, FIG. 9 b). In order to ensure proper vesicular localization, weused full-length Syb as the linker between CFP and YFP. Because CFP isrelatively resistant to the acidic environment in the vesicle lumen(Tsien, The green fluorescent protein. Annu. Rev Biochem 67, 509-544(1998), the CFP was fused to the C-terminus of Syb and is predicted toreside inside vesicles, while the YFP was fused to the N-terminus of Syband faces the cytosol (FIG. 9 b). This sensor is denoted asYFP-Syb(FL)-CFP. Since FRET is unlikely to occur between CFP and YFPhere, a novel approach was taken to monitor the cleavage by BoNT/B.Cleavage of YFP-Syb(FL)-CFP sensor would generate two fragments,including a N-terminal portion tagged with YFP, which would be releasedinto the cytosol, and a short C-terminal portion tagged with CFP that isrestricted inside vesicles. Thus, toxin activity would result in theredistribution of YFP fluorescence in cells. YFP-Syb(FL)-CFP proved tobe an efficient toxin sensor in cells. As indicated in FIG. 9 c,treatment with BoNT/B resulted in the dissociation of YFP from thevesicles and its redistribution into the cytosol. We note that thesoluble YFP fragment was able to enter the nucleus, where there was nofluorescence signal prior to toxin treatment (FIG. 9 c), providing anarea where the YFP redistribution can be readily detected. Unlike theFRET assay, this detection method does not require a short distancebetween CFP and YFP, thus providing a novel approach to monitor proteaseactivity in living cells.

To exclude the possibility that the inefficient cleavage of the sensorcontaining Syb(33-94) fragment is due to the lack of the N-terminal 32amino acid, a sensor containing the truncated form of Syb that lacks theN-terminal 32 residues (denoted as CFP-Syb(33-116)-YFP) was built. Thissensor contains the same cytosolic domain of Syb with the inefficientsensor (residues 33-94), plus the transmembrane domain (residues95-116), which anchors it to vesicles. When assayed in parallel,significant amount of CFP-Syb(33-116)-YFP was cleaved by BoNT/B after 48hours, while there was no detectable cleavage of CFP-Syb(33-94)-YFP(FIG. 9 d), indicating the vesicular localization determines thecleavage efficiency in cells. This conclusion is further supported by arecent report that the presence of negatively charged lipid mixturesenhanced the cleavage rate of Syb by BoNT/B, TeNT, and BoNT/F in vitro(Caccin et al., VAMP/synaptobrevin cleavage by tetanus and botulinumneurotoxins is strongly enhanced by acidic liposomes. FEBS Lett. 542,132-136 (2003). It is possible that toxins may favor binding tovesicular membranes in cells, thus increasing the chance to encounterSyb localized on vesicles. Alternatively, it is also possible that thepresence of the transmembrane domain may be critical for maintaining aproper conformational state of Syb that is required for efficientcleavage.

Using full length SNAP-25 and Syb II as the linkers provided excellentoptical reporters that can mirror endogenous substrate cleavage inliving cells. These two reporters should be able to detect all sevenbotulinum neurotoxins and tetanus neurotoxin (TeNT). The substratelinker sequence can be readily modified to achieve specific detectionfor individual BoNTs or TeNT by changing the length or mutating othertoxin cleavage or recognition sites. These toxin biosensors shouldenable the cell-based screening of toxin inhibitors, and the study oftoxin substrate recognition and cleavage in cells.

The foregoing description and examples have been set forth merely toillustrate the invention and are not intended to be limiting. Sincemodifications of the disclosed embodiments incorporating the spirit andsubstance of the invention may occur to persons skilled in the art, theinvention should be construed broadly to include all variations fallingwithin the scope of the appended claims and equivalents thereof. Allreferences cited hereinabove and/or listed below are hereby expresslyincorporated by reference.

What is claimed is:
 1. An isolated polynucleotide molecule encoding amolecular construct, wherein the construct is capable of anchoring to aplasma membrane or a vesicular membrane of a cell, the constructcomprising (1) a linker peptide, (2) a FRET pair comprising a donorfluorophore moiety and an acceptor fluorophore moiety, and (3) amembrane-anchoring domain that directs the anchoring of the construct tothe plasma or vesicular membrane of the cell, wherein the linker peptidecomprises a cleavage recognition site of a botulinum neurotoxin selectedfrom the group consisting of synaptobrevin, syntaxin and SNAP-25, andwherein the membrane-anchoring domain comprises a fragment that containsthe palmitoylation site (residues 83-120) of SEQ ID NO: 7, or thetransmembrane domain (residues 95-116) of SEQ ID NO:
 8. 2. An expressionvector comprising a polynucleotide molecule of claim 1, operably linkedto a promoter.
 3. An expression vector of claim 2, wherein the promoteris an inducible promoter.
 4. An isolated cell comprises an isolatedpolynucleotide molecule of claim
 1. 5. A cell of claim 4, wherein thecell is selected from the group consisting of a primary cultured neuroncell, PC12 cell or a derivative thereof, a primary cultured chromaphincell, a neuroblastoma cell, a human adrenergic SK-N-SH cell, and a NS-26cell line.
 6. A cell of claim 5, wherein the cell is a cortical neuroncell, a hippocampal neuron cell, a spinal cord motor neuron cell, or amurine cholinergic Neuro 2a cell.
 7. An isolated polynucleotide of claim1, wherein the donor fluorophore moiety is a green fluorescent protein(GFP) or a variant thereof, and the acceptor fluorophore moiety is acorresponding variant of the green fluorescent protein.
 8. The isolatedpolynucleotide of claim 1, wherein the palmitoylation site is fused tothe N-terminus of CFP-SNAP-25(141-206)-YFP.
 9. The isolatedpolynucleotide of claim 1, wherein the construct is CFP-SNAP-25(FL)-YFP,YFP-Syb(FL)-CFP, or CFP-Syb(33-116)-YFP, wherein, where SNAP-25(FL) isSEQ ID NO:7, Syb(FL) is SEQ ID NO:8, and Syb(33-116) is SEQ ID NO:10.10. The isolated polynucleotide of claim 1, wherein the construct isCFP-SNAP-25(141-206)-YFP, with a fragment of SNAP-25 that contains thepalmitoylation site (residues 83-120) fused to its N-terminus, whereinSNAP-25(141-206) is SEQ ID NO:9, and the palmitoylation site (residues83-120) is SEQ ID NO:11.
 11. The isolated polynucleotide of claim 1,wherein at least one of the fluorophores is selected from the groupconsisting of a green fluorescent protein and a variant thereof.
 12. Theisolated polynucleotide of claim 1, wherein one of the fluorophores isselected from the group consisting of a yellow fluorescent protein and avariant thereof.
 13. The isolated polynucleotide of claim 1, wherein theFRET pair comprises a yellow fluorescent protein (YFP) and a redfluorescent pair (CFP).